The present invention is generally in the area of improved solutions containing arabinogalactan for washing and separating sperm.
Sperm must be washed prior to use in most diagnostic or research protocols, such as infertility testing, in vitro fertilization and freezing. Washing is done to limit damage to the sperm cell by components of seminal plasma, such as antibodies, white blood cells, red blood cells and bacteria. Washing also removes dying or dead sperm which release enzymes and other products which are toxic to healthy sperm.
Generally, sperm are separated by allowing the motile sperm to swim away from the debris (sperm swim-up), by centrifuging the sperm through a gradient and collecting a pellet of live sperm (washing), or by passing the sperm through a column that binds the dead or unhealthy sperm. Each of these current techniques has its own disadvantages. Swim-up only recovers a small number of the normal sperm. Column methods have poor selectivity for the normal sperm in the ejaculate. Centrifugation through a density gradient results in high sperm recovery but also can produce products which are toxic to sperm, so that an additional wash step is required to remove the products. Centrifugation also can produce sperm with reduced motility and fertility during in vitro fertilization. Parrish et al., Theriogenology, 44:859-869 (1995); and Tanphaichitr et al., Gamete Research, 20:67-81 (1988).
Methods and compositions for washing and separating sperm have been the subject of many studies. Trounson and Gardner, Handbook of In Vitro Fertilization, CRC Press, Boca Raton, 1994, pp. 46-50. Bongso et al., Fertility and Sterility, 51:850-854 (1989) discloses a study of the improvement of concentration and motility of sperm following separation in Ficoll, a synthetic polymer of sucrose. U.S. Pat. No. 4,007,087 to Ericsson discloses fractionating sperm in a medium including soluble materials such as proteins, peptides and dextran. U.S. Pat. No. 4,327,177 to Shrimpton discloses the separation of sperm by density in a nutrient media derived from mammalian milk. U.S. Pat. No. 4,605,558 to Shrimpton discloses a method of separating X and Y sperm in a density gradient and an osmolality gradient in a medium derived from milk. Platov et al., Ovtsevodstvo, 10:38-39 (1980) (Abstract) discloses the use of gum arabic, cherry resin and apricot resin in a sheep semen freezing medium.
Arabinogalactan is a water-soluble polysaccharide which can be isolated from trees of the genus Larix, particularly Larix occidentalis Nuttall (western larch). Methods for the preparation of ultrarefined arabinogalactan are disclosed in U.S. Pat. No. 5,116,969. Hill et al., J. Lab. Clin. Med., 111:73-83 (1988) discloses the use of arabinogalactan to obtain washed murine platelets by centrifugation. The preparation of arabinogalactan derivatives and fragments is described in Prescott, et al., Carbohydrate Research, 278:113-128 (1995); and U.S. Pat. No. 5,478,576 to Jung, et al., the disclosures of which are incorporated herein by reference.
There is a need for the development of improved products for washing and separating sperm for protocols such as infertility testing, in vitro fertilization, and freezing.
It is therefore an object of the invention to provide solutions for washing and separating sperm to produce sperm samples with optimal motility and viability, and which can be used in a variety of diagnostic and research applications, such as fertility testing, and in vitro fertilization.